Multistage process for the preparation of highly pure deferoxamine mesylate salt

ABSTRACT

The present invention provides a purification process whereby deferoxamine B produced by a microorganism and in mixture with other polyhydroxamates produced by the microorganism may be converted into its mesylate salt substantially free of the other polyhydroxamates and substantially free of chloride ion. The process includes adsorption and desorption of the deferoxamine B on an adsorption resin, direct precipitation of the deferoxamine free base out of the eluent from the adsorption resin, contacting of the deferoxamine B free base with methanesulfonic acid and isolation of the deferoxamine B mesylate salt by precipitation. This process minimizes decomposition of deferoxamine B.

RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119 of HungarianPatent Application P 99 04454, filed Dec. 1, 1999.

FIELD OF THE INVENTION

This invention relates to a process for preparing highly puredeferoxamine B mesylate.

BACKGROUND OF THE INVENTION

Deferoxamine B, represented by formula I, is a polyhydroxamate ironchelator that is useful for reducing iron concentration in human bloodplasma.

The systematic chemical name of deferoxamine B (also known asdeferriferrioxamine B) isN′-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-1,4-dioxobutyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-N-hydroxy-butanediamide.Deferoxamine B has the desirable property of high affinity for ferriciron (Ka=10³¹) coupled with a very low affinity for calcium (Ka=10²).Goodman and Gilman, The Pharmacological Basis of Therapeutics 1668 (9thed. 1996).

Deferoxamine B is indicated for treatment of acute iron intoxication andchronic iron overload due to transfusion dependant anemias. It promotesiron excretion in patients with secondary iron overload from multipletransfusions, as may occur with treatment of some chronic anemias suchas thalassemia. Long term therapy slows accumulation of hepatic iron andretards or eliminates progression of hepatic fibrosis. Physicians DeskReference 2010 (54th ed. 1999). Deferoxamine B is not well-absorbedorally; it must be administered parenterally.

Industrial scale fermentation processes for producing deferoxamine B usethe Streptomyces pilosus bacteria strain, which produces a variety ofpolyhydroxamate compounds, but predominantly deferoxamine B, in aculture medium poor in iron. Belgian Patent No. 619,532. Deferoxamine Bis typically isolated from the fermentation broth as its hydrochloridesalt. The hydrochloride salt is not pharmaceutically acceptable forparenteral administration to humans. Therefore, it must be convertedinto a pharmaceutically acceptable salt. The mesylate salt is the FDAapproved deferoxamine salt. The U.S. Pharmacopeia & National Formularydirects that pharmaceutical grade deferoxamine B mesylate contain notmore than 120 ppm chloride. USP/NF 24/19, pp. 499-500 (1999). This hasproven to be a challenging standard to meet and there remains a need foran improved process for transforming deferoxamine B produced byfermentation into a pharmaceutically acceptable pure mesylate salt foradministration to patients.

Belgian patent 619,532 discloses purification of deferoxamine B obtainedvia fermentation using adsorption chromatography. Activated carbon,activated diatomaceous earth (e.g. fuller's earth) or ion-exchange resin(Asmit) are recommended adsorbents. Alternative adsorbents are said toinclude aluminum oxide, magnesium silicates, silica gel, gypsum andion-exchange resins. According to the Belgian 619,532 patent,deferoxamine B may be eluted using a mobile phase of methanol-water,pyridine-water or acetic acid-methanol.

International Publication No. WO 93/09088 and European patent 347,163describe chromatography over silica gel as a method for purifyingdeferoxamine B produced by synthetic means, not by microbiologicalmeans.

International Publication No. WO 93/03045 describes purification of ironchelate complexes of deferoxamine B and structurally related compoundsusing a polystyrene adsorption resin.

U.S. Pat. Nos. 3,153,621 and 3,118,823 disclose partial purification ofdeferoxamine B using ion-exchange resins. It is believed that theteachings of these patents lead to a mixture of deferoxamine B withother polyhydroxamates that are produced by the Streptomyces pilosusstrain.

Belgian Patent 616,139 discloses that deferoxamine B mesylate salt canbe obtained from the deferoxamine B hydrochloride salt by passing anaqueous solution of deferoxamine hydrochloride over Dowex-1, X-16 anionexchange resin (in OH⁻ form), adding methanesulfonic acid in equivalentquantity to the resulting deferoxamine base in aqueous solution, thenevaporating the water and, lastly, purifying the deferoxamine mesylatesalt by recrystallization from aqueous alcohol or a mixture ofwater-methanol-acetone.

U.S. Pat. No. 5,374,771 describes purification of crude deferoxamine Bhydrochloride by ion-exchange chromatography and multiplerecrystallizations. The mesylate salt is prepared directly from thepurified deferoxamine B hydrochloride by contacting with an anionexchange resin having mesylate counter-ion. Deferoxamine B mesylate isobtained from the aqueous solution by lyophilization.

Bickel, H. et al., Helvetica Chimica Acta, 1385-1389 (1963), describespurification of deferoxamine B base by multiple recrystallizations froma water-alcohol mixture. The deferoxamine B base is prepared by anionexchange, evaporation to dryness, and multiple recrystallizations. Thedeferoxamine B base is then suspended in a water-methanol mixture and amineral acid salt prepared. Subsequently the deferoxamine B solution isevaporated and the residue is recrystallized from a water-methanolmixture.

Removal of chloride ion from an aqueous solution of deferoxamine B isone of the steps in each of the methods described above for purifyingdeferoxamine B from a fermentation broth. In each case, the chloride ionis removed using an anion exchange resin. However, ion-exchange resinsalone are not effective for isolating deferoxamine B mesylate free ofchloride ion as is required in order to achieve a pharmaceuticallyacceptable state of purity.

The above-mentioned purification methods that use silica gel or aluminumoxide as adsorbents are inefficient, time consuming and expensive. Theother conventional substitutes—activated charcoal, diatomaceous earth,magnesium silicates, and gypsum—are poor adsorbents of deferoxamine. Forthese reasons, there remains a need in the art for a rapid, efficientand inexpensive method for obtaining deferoxamine B mesylate from afermentation broth free of chloride ion, or at least free of chlorideanion in an amount greater than 120 ppm.

Furthermore, the above-described methods do not efficiently removefermentation products that are structurally related to deferoxamine B,which must be removed before use of the deferoxamine B in apharmaceutical product. Typically, an extract from adeferoxamine-producing fermentation broth contains, relative to thedeferoxamine B content, approximately 6 to 20 mole % polyhydroxamatecompounds that are structurally related to deferoxamine B. Suchcompounds include other deferoxamines, such as deferoxamine A, C, D₁,D₂, E, F and G. Since deferoxamine B and the other deferoxamines havesimilar chemical properties, none of the known purification processes orcombinations thereof have been able to reduce the quantity of theimpurities below about 2.5%.

In addition to their uncertain therapeutic, and possibly toxic effect itis important to remove these impurities in order to accurately determinethe deferoxamine B concentration in a solution, such as a sterilesolution for injection. The USP/NF specifies that a deferoxamine Binjection solution contain between 90.0 and 110% of the labeledconcentration. USP/NF 24/19, p. 500 (1999) The assay for determiningconcentration specified by the USP/NF is a photometric absorbenceintensity measurement of the iron complex at 485 nm. Id. Thestructurally related impurities also form complexes which absorb in the485 nm range, leading to an overestimation of the deferoxamine Bcontent. We have found that a photometric assay of a solution ofdeferoxamine B mesylate obtained by fermentation according to methodsknown in the art overestimates the concentration of deferoxamine B byabout 3%. Thus, there remains a need in the art for a rapid, efficientand inexpensive method for obtaining deferoxamine B mesylate from afermentation broth free of other polyhydroxamates formed by themetabolic processes of Streptomyces (e.g. pilosus or 101/87) as well asfree of chloride ion.

SUMMARY OF THE INVENTION

The need for high purity in drugs in order to prevent undesirableeffects caused by impurities and the need for accuracy in determiningthe potency of a deferoxamine B mesylate drug are both met by thepresent invention. In the course of our experiments with deferoxamine B,we found that fermentation byproducts that are chemically unrelated todeferoxamine B and related substances with significantly differentpolarity could be removed by adsorption chromatography, but that otherpolyhydroxamates could not. We have found that the most effective meansof reducing the quantity of other polyhydroxamates is by precipitationof the deferoxamine B free base after preliminary cleanup by adsorptionchromatography. We also found that decomposition products are formed ifdeferoxamine B is evaporated to dryness. Deferoxamine B is a reasonablystable solid, but it is prone to decomposition in concentrated solution.As a dilute solution of deferoxamine B is evaporated, the solutionconcentration rises, which causes decomposition and a solidified productof low purity. We found that this decomposition can be minimized byconcentration- and pH-adjustment of the eluent from the adsorption resinwithin certain parameters and then direct precipitation of deferoxamineB free base from the eluent.

Thus, we have discovered that in order to efficiently remove bothchloride and polyhydroxamate impurities, to minimize the formation ofdecomposition products, and to separate them to the extent that theirformation is unavoidable, that one should apply, in succession,chromatography on adsorption resin, precipitation of the deferoxamine Bfree base from a mixture of water and a water-soluble organic solvent,formation of the deferoxamine mesylate salt and crystallization of thatsalt from a mixture of water and a mesylate salt anti-solvent or from amixture of methanol and a mesylate salt anti-solvent. This processproduces deferoxamine B mesylate containing less than 2.5 mole % ofother polyhydroxamate impurities and less than 90 ppm chloride ion.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a purification process for production ofhighly pure deferoxamine B mesylate from a source material containingdeferoxamine B produced by a microbiological process. The purificationprocess is feasible on an industrial scale and is highly economicalcompared to other known purification processes.

Microbiological processes for producing deferoxamine B are known in theart, such as the method described in U.S. Pat. No. 3,158,552, which isherein incorporated by reference in its entirety. As described in the'552 patent, Streptomyces pilosus is cultivated in a submersion mediumwith a low iron content to stimulate production of deferoxamines whichthe microorganism uses to extract iron from its environment. At the endof the fermentation, deferoxamine B is freed from complexation with Fe³⁺ions in the culture medium by addition of a competitive iron chelatingagent, 8-hydroxyquinoline. The broth is then filtered to remove cellmass and an aqueous solution containing deferoxamine B is obtained.After further processing, deferoxamine B hydrochloride, in mixture withother deferoxamines, is precipitated from aqueous solution.

The process of the present invention may be applied to a source materialobtained from a fermentation broth after processing such asconcentration or extraction as described in the '552 patent or afterprocessing as described in U.S. Pat. No. 5,374,771. Likewise, anevaporated fermentation broth extract containing deferoxamine B may betaken up in either aqueous or organic solvent and used as sourcematerial for deferoxamine B. Preferably, the source material ofdeferoxamine B is an aqueous solution with a deferoxamine Bconcentration of from about 5 to about 70 g L⁻¹, more preferably fromabout 10 to about 30 g L⁻¹. The purification process is furtherdescribed as it is applied to such an aqueous solution of deferoxamine B(hereafter “the crude deferoxamine B solution”).

The purification process of the present invention comprises threestages. First, impurities chemically unrelated to deferoxamine B andrelated substances with significantly different polarity are removed bychromatography over an adsorption resin. In the second stage,deferoxamine B is separated from chloride anion and otherpolyhydroxamates by precipitation of the deferoxamine B as its freebase. In the third stage, the deferoxamine B, in free base form, issuspended in a mixed solvent, treated with methanesulfonic acid todissolve the free base and crystallized as its mesylate salt.

Chromatographic Separation of Impurities

In the first stage of the invention, compounds that are chemicallydissimilar to deferoxamine B and related substances with significantlydifferent polarity are substantially separated by contacting thedeferoxamine B source material with a bed of adsorption resin andcollecting deferoxamine B in solution as an eluent from the bed.Preferred adsorption resins are unsubstituted and substituted aromatictype resins, aromatic resins with hydrophobic groups, acrylic andmethacrylic resins. Especially preferred adsorption resins includeDiaion® resins of the FP, HP, SP and HPMG series (Mitsubishi ChemicalCorp.) and Amberlite® resins of the XAD series (Rohm & Haas), the mostpreferred being Diaion® SP 207 and Amberlite® XAD 1180. Deferoxamine Bcan be eluted from these resins with a mixture of water and awater-soluble organic solvent such as methanol, ethanol, acetonitrileand tetrahydrofuran.

A preferred chromatographic procedure uses a pre-column and a maincolumn containing separate beds of adsorption resin. According to thispreferred procedure, the crude deferoxamine B solution is first elutedthrough the pre-column which contains a small amount of adsorptionresin, i.e. about 2 to 6 one hundredths of the volume of the crudedeferoxamine B solution. The pre-column may be mounted atop the maincolumn and the crude deferoxamine B solution may be allowed to passthrough the pre-column without eluting with solvent because only a minorportion of the deferoxamine B is adsorbed on the pre-column.Alternatively, the pre-column may be eluted with solvent. The eluent maybe a mixture of water and a water-soluble organic solvent as describedabove, but preferably, for improved retention of deferoxamine B on themain column, the elution solvent, if used, is salt water as describedbelow. A pre-column may result in a small reduction in yield ofdeferoxamine B mesylate at the end of the process but its use has theadvantage of higher ultimate purity and the economic advantage ofgreater ease of regeneration of the adsorption resin of the main column.

After optionally passing the crude deferoxamine B solution through apre-column, the solution is preferably treated with an inorganic salt toenhance adsorption of deferoxamine B on the main column. Chloride saltsand sulfate salts are preferred inorganic salts, the most preferredbeing ammonium chloride and ammonium sulfate. The inorganic salt shouldbe added in amount of about 2 to about 15 g L⁻¹ of solution, morepreferably about 5-10 g L⁻¹. The crude deferoxamine B solution is thenloaded onto a main column containing a bed of absorption resin whosevolume is from about {fraction (1/20)}th the volume of the crudedeferoxamine B solution to about the same volume as that of the crudedeferoxamine B solution. Preferably, the volume of the bed of absorptionresin is from about ¼ to about ¾ the volume of the crude deferoxamine Bsolution. A more highly concentrated crude deferoxamine B solutiongenerally requires less resin than does a more dilute solution Thedeferoxamine B adsorbs onto the resin and the aqueous solution depletedof deferoxamine B is either allowed to drain from the bed or is drivenfrom the bed with a solution of the inorganic salt. Partially purifieddeferoxamine B is then recovered from the adsorption resin by elutingwith a mixture of water and a water-soluble organic solvent such asmethanol, ethanol, acetonitrile or tetrahydrofuran, most preferablyacetonitrile. Generally, the proportion of organic solvent in themixture should be from about 1% to about 70% (v/v), preferably about 1%to about 50% (v/v), depending on the resin. In particular, an 88:12:3water:acetonitrile:methanol mixture is a very good solvent mixture foreluting deferoxamine B from Amberlite® XAD 1180 resin.

Gradient elution may also be advantageously used to elute deferoxamine Bfrom any of the suitable resins. One such generally applicable gradientmethod involves eluting first with salt water, then with watercontaining an organic elution solvent. Eluting first with water, thenwith either a water-acetonitrile or water-methanol mixture, optionallyfollowed by elution with a water-ethanol or water-acetone, gives goodresults. A particularly preferred gradient method for elutingdeferoxamine B from Amberlite® XAD 1180 resin is to elute first withsalt water, then with a 90:10 mixture of water-acetonitrile, and thengradually increasing the acetonitrile content to an 80:20water-acetonitrile mixture.

Whether gradient or non-gradient elution is used, deferoxamine B will becollected from the main column dissolved in aqueous organic eluentcontained in one or more fractions cut from the eluent stream. Thedeferoxamine B-containing eluent may optionally be decolorized such asby treatment with activated carbon or a cationic exchange resin likeAmberlite® IRC 50, Duolite® C467 and Lewatit® CNP 80 according tomethods known in the art. If the deferoxamine B is collected in three ormore fractions—an early eluting fraction, one or more middle elutingfractions, and a late eluting fraction—then the middle fraction(s)containing deferoxamine B will typically be colorless. Only the earlyeluting and late eluting fractions are likely to have coloration.Accordingly, the desirability of a decolorizing treatment will depend onhow the fractions are cut. If both early eluting and late elutingfractions are collected and combined with the middle fraction(s), it isdesirable to use both activated carbon and a cation exchange resin todecolorize since the early-eluting and late-eluting fractions responddifferently to different decolorizing methods.

Precipitation of Deferoxamine B Free Base

In the second stage of the inventive process, deferoxamine B free baseis precipitated out of the eluent from the adsorption resin. Althoughthe deferoxamine B concentration in the eluent may vary greatlydepending upon the solvent mixture(s) used to elute deferoxamine B fromthe resin, it is not necessary to evaporate the eluent to dryness inorder to establish an appropriate concentration and solvent compositionfor precipitating the base. Evaporation of the eluent to dryness is, infact, disadvantageous because of the instability of the hydroxamategroups of deferoxamine B toward high solution concentration and appliedheat. Rather, the eluent may be prepared for precipitation of the freebase in high yield and high purity by the following the procedure.

If the deferoxamine B concentration of the eluent is lower than about 50g L⁻¹, the eluent should be evaporated under mild conditions to adeferoxamine B concentration of about 50 g L⁻¹ to about 150 g L⁻¹,preferably about 80 g L⁻¹ to about 100 g L⁻¹ and most preferably about90 g L⁻¹. The preferred organic elution solvents listed above are lowerboiling than water and in some cases azeotrope with a minor amount ofwater, so the proportion of water in the solvent mixture increases uponconcentration. The concentrated eluent is then diluted with acetonitrilein an amount of from about 0.5 to about 1.5 times the volume of theconcentrated eluent. Addition of acetonitrile at this stage was found toprevent precipitation of deferoxamine B hydrochloride (or sulfate, asthe case may be depending upon the inorganic salt used).

The concentration-adjusted eluent containing deferoxamine B in water andacetonitrile (and optionally another organic elution solvent) is thenpH-adjusted to between about 8.6 and 10.5, more preferably between about9.4 and about 10.0 pH. The pH can be adjusted using either a basic ionexchange resin or by addition of an aqueous alkaline solution or both.Suitable basic ion exchange resins include Amberlite® resins of the IRAseries and Diaion® resins of the WK series, the most preferred beingAmberlite® IRA 67 in OH⁻ form. Suitable alkaline solutions are solutionsof NaOH, KOH, ammonia or an amine, the most preferred being concentratedaqueous ammonia.

In an especially preferred pH-adjustment technique, the pH of theconcentration-adjusted eluent is first tested. If it is below pH 8.0,the pH is adjusted to between about 8.0 and 9.3 with a basic ionexchange resin such as Amberlite® IRA 67 in OH⁻ form. The resin is thenseparated and aqueous ammonia is added until the concentration-adjustedeluent reaches a pH of between about 9.4 and 10.0.

After pH-adjustment, crystallization of the deferoxamine B free base isinduced by addition of a deferoxamine B free base anti-solvent such asacetonitrile, ethanol, methanol, or acetone. Preferred deferoxamine Bfree base anti-solvents are acetonitrile and acetonitrile-acetonemixtures. As used herein, the term “anti-solvent” means a liquid inwhich a compound is poorly soluble. Thus the term anti-solvent relatesto the solubility of a particular compound in that liquid. The presentinvention uses two classes of anti-solvent: deferoxamine B free baseanti-solvents and deferoxamine B mesylate anti-solvents.

The rate of addition of deferoxamine B free base anti-solvent to thepH-adjusted eluent is not critical, but a slower addition rate tends toproduce larger crystals and a purer crystalline deferoxamine B freebase. On a production scale, a practical rate of addition is about 0.5to about 2 times the volume of pH-adjusted eluent per hour, thoughincreased crystal size and higher purity may be realized with a sloweraddition rate. The total amount of added deferoxamine B free baseanti-solvent is preferably about 1.5 to 10 times, more preferably about2 to 8 times and most preferably about 2.5 to 5 times the volume of thepH-adjusted eluent. The temperature of the solution may be maintained atanywhere from about −20° C. to about 40° C. during the crystallization,although preferably the temperature is maintained at between about 0° C.and about 20° C.

After crystallization is complete, the deferoxamine B free base crystalsmay be isolated by any conventional means, such as filtration ordecantation. Optionally, the crystals of the free base may be furtherpurified by recrystallization. Acetonitrile-water mixtures are also goodsolvent systems for recrystallization.

Crystallization of Deferoxamine B Mesylate

In the third stage of the inventive process, the crystallized free baseis suspended in a mixed solvent. Methanesulfonic acid is then added tothe suspension which causes the deferoxamine B free base to go intosolution. After complete dissolution of the deferoxamine B free base,addition of methanesulfonic acid is continued until the solution reachesa pH of about 3 to about 6, more preferably about 3.5-4.5. DeferoxamineB mesylate is then allowed to precipitate.

Suitable mixed solvents for suspending the deferoxamine B free base aremixtures of a deferoxamine B mesylate solvent, either water or methanol,and a deferoxamine B mesylate anti-solvent. Deferoxamine B mesylateanti-solvents include C₁-C₇ aliphatic alcohols, acetone, methyl formate,methyl acetate, ethyl acetate, hexane, toluene, tetrahydrofuran andacetonitrile. The polar, solvent component of the mixed solvent, i.e.the methanol or the water, is preferably mixed in a ratio of about 1:1to about 1:10 with the anti-solvent component, more preferably in aratio of about 1:5. Preferred mixed solvents are water-ethanol mixtures,water-acetonitrile mixtures, methanol-ethanol mixtures andmethanol-acetonitrile mixtures, the most preferred being 1:5water-ethanol mixtures and 1:3 methanol-ethanol mixtures. The volume ofmixed solvent that should be used to suspend the free base is from about5 to about 20 L per kilogram of deferoxamine B free base, morepreferably from about 7 to about 15 L per kilogram.

After forming the suspension, methanesulfonic acid is added to thesuspension. The amount of methanesulfonic acid that is required can beapproximated by calculating the amount required to provide oneequivalent of the acid to the base. Alternatively, the amount ofmethanesulfonic acid necessary to provide one equivalent may beaccurately gauged by observing the amount of undissolved free base thatremains suspended during addition of the acid. The methanesulfonic acidshould be added slowly for this purpose since the rate at which thesuspended base can react with the acid is rate limited by the surfacearea of the crystals. After the all of the deferoxamine B free base hasdissolved, the pH of the solution should be monitored, while additionalmethanesulfonic acid is added to bring the pH of the solution to betweenabout 3 and about 6, preferably about 3.5-4.5. After the desired pH isreached, deferoxamine B mesylate crystals are allowed to precipitate.

To accelerate precipitation, the solution may be cooled to about −20 toabout 10° C. In addition, precipitation may be accelerated by addingmore deferoxamine B mesylate anti-solvent to the mixture, i.e. by addinga C₁-C₇ aliphatic alcohol, acetone, methyl formate, methyl acetate,ethyl acetate, hexane, toluene, tetrahydrofuran or acetonitrile. Afterprecipitation is complete deferoxamine B mesylate is isolated byconventional means such as filtration or decantation.

Having thus described the inventive process with reference to itspreferred embodiments, the invention will now be further illustrated bythe examples which follow.

EXAMPLES General

HPLC Conditions (Reverse phase): column: C₁₈, particle size 10μ, length250 mm, diameter 4.6 mm; mobile phase: 5.5% THF/water, 0.13% (NH₄)H₂PO₄(w/v), 0.04% sodium edetate (w/v); flow rate: 2 ml min.⁻¹ ; detection:UV λ=220 nm.

Example 1

Crude deferoxamine B hydrochloride (7.88 kg) containing 5.30 kgdeferoxamine B was dissolved in water (362 L). The aqueous solution wasthen passed through an equal volume of Diaion® SP 207 adsorption resin(Mitsubishi Chemical Corp.) in a 10 liter chromatographic column at aflow rate of 14 L h⁻¹. The eluent contained 5.04 kg deferoxamine.Ammonium chloride was then added in an amount of 5 g L⁻¹ to the eluentwith stirring until the ammonium chloride completely dissolved. Thesolution was then loaded onto a column containing Amberlite® XAD 1180adsorption resin (132 L) (Rohm & Haas) and driven into the bed with saltwater. The column was eluted with 10% acetonitrile-water and then 20%acetonitrile-water at a flow rate of 14 L h⁻¹. The eluent was collectedin fractions. The main fraction yielded deferoxamine hydrochloridesolution containing 4.53 kg of deferoxamine B. The main fraction wasthen decolorized over activated carbon (45 g) and Duolite® C 467 (Rohmand Haas) ion exchange resin in H⁺ form yielding deferoxaminehydrochloride solution containing 4.03 kg of deferoxamine B.

The decolorized eluent was evaporated to a deferoxamine B concentrationof 90 g L⁻¹. A volume of acetonitrile equal to that of the concentratedeluent was then added and the basicity was adjusted by addition ofAmberlite® IRA 67 (Rohm and Haas) in OH⁻¹ form to a pH between 8.0149.3. After pH-adjustment with the anion exchange resin, the pH wasraised to between 9.4 and 10.0 with aqueous ammonia. Deferoxamine freebase was then precipitated by adding two volumes of acetonitrile to thesolution and cooling to −5° C. Crystallization was complete afterseveral hours. The crystals of deferoxamine free base were isolated byfiltration and then suspended in 1:1 acetonitrile-water, filtered again,washed with acetonitrile and dried to yield 3.81 kg of deferoxamine freebase. HPLC chromatography showed that the free base was 97% pure.Following the USP chemical test <221> for determining chloride content,the precipitated deferoxamine B free base was found to have a chloridecontent of less than 60 ppm. USP/NF24/19, pp. 1857-1858 (1999)

The deferoxamine free base was suspended in a 14:3 mixture of ethanoland water (42.5 L). Dilute methanesulfonic acid was slowly added to thesuspension until the free base dissolved and the solution attained a pHof between 3.7 and 5. Ethanol (200 L) was added to the solution toprecipitate the mesylate salt and the resulting suspension was cooled to−10° C. and maintained at −10° C. for several hours. The resultingcrystals were isolated by filtration, washed with ethanol (50 L) anddried under vacuum to yield 3.4 kg of deferoxamine mesylate. HPLCchromatography showed that the mesylate contained 1.84 weight %impurities. The moisture content as determined by Karl-Fischer Analysiswas 0.2 mole %. USP/NF 24/19, pp. 2003-2004 (1999). Coloration wasbetter than Y5 as measured according to the European Pharmacopeiacriteria for grading coloration.

Example 2

Crude deferoxamine B hydrochloride (707 g) containing 587 g deferoxamineB was dissolved in water (10 L). The aqueous solution was passed througha chromatography column containing Diaion® SP 207 adsorption resin (1 L)at a flow rate of 0.35 L h⁻¹. The eluent from the column was found tocontain 562 g of deferoxamine B. The eluent was then loaded onto a bedof Amberlite® XAD 1180 adsorption resin (7.2 L) in a chromatographycolumn. The bed was eluted at a flow rate of 0.7 L h⁻¹, first with 5.6 Lof a 10 g L⁻¹ solution of ammonium chloride in water and then with a 1:4methanol-water mixture. Deferoxamine B (432 g) was collected in a singlefraction. This fraction was decolorized by stirring over activatedcharcoal (40 g) and filtered. The decolorized eluent was evaporated to adeferoxamine B concentration of 60 g L⁻¹.

An equal volume of acetonitrile was then added. Concentrated aqueousammonia was then added until the solution attained a pH of 9.8. Then,four volumes of acetonitrile were added to precipitate the deferoxamineB free base. The resulting suspension was cooled to −4° C. andmaintained at reduced temperature for 24 h, after which time thecrystals were isolated by filtration. The isolated crystals were thenwashed by three repetitions of suspending in 1:1 water-acetonitrile andfiltering. The crystals were then dried at 40° C. at ambient pressure toyield the deferoxamine B free base (341 g) in 95.2% purity (w/w) asdetermined by HPLC and with a chloride content of less than 60 ppm.

The deferoxamine B base was then suspended in a mixture of ethanol (3.1L) and water (0.46 L). Methanesulfonic acid was slowly added until thedeferoxamine B base dissolved and the solution reached a pH of 3.5.Another 30 L of ethanol was added to the solution to precipitatedeferoxamine B mesylate. The resulting suspension was then cooled tobetween −5° C. and 0° C. and maintained at reduced temperature for 24 h.The crystals were isolated by filtration, washed with ethanol and driedunder vacuum to yield deferoxamine B mesylate (299 g) with a purity of98% (w/w) as determined by HPLC analysis.

Example 3

Crude deferoxamine B hydrochloride (760 g) containing 530 g ofdeferoxamine B was dissolved in 1:1 acetonitrile-water ( 14 L) at 40° C.The resulting solution was then filtered and pH-adjusted to 9.9 byaddition of concentration aqueous ammonia. Acetonitrile (49 L) was thenadded to precipitate the deferoxamine B free base. The resultingsuspension was then cooled to 17° C. and maintained at reducedtemperature for four hours. The precipitate was isolated by filtration,washed with acetonitrile and dried under reduced pressure to yielddeferoxamine B free base (403 g). The base was then suspended in water(20 L) and hydrochloric acid was slowly added until the base dissolvedand the resulting solution reached a pH of between 5.0 and 5.5.

The solution was loaded onto a bed of Amberlite® XAD 1180 adsorptionresin (7.2 L) in a chromatography column and eluted with 25%acetonitrile-water. The deferoxamine B (320 g) was obtained in a singlefraction. The deferoxamine B-containing eluent was decolorized overactivated charcoal (10 g). After filtration, the decolorized eluentcontaining 305 g of deferoxamine B was concentrated to about 110 g L⁻¹.An equal volume of acetonitrile then was added to the solution. The pHof the solution was then adjusted to between 8.6 and 9.0 by addingAmberlite® IRA 67 anion exchange resin in OH⁻¹ form. Aqueous ammonia wasadded to the solution to precipitate deferoxamine B free base. Theaddition was continued until the suspension reached pH 9.8. Thesuspension was then cooled to −4° C. and maintained at reducedtemperature for several hours. Crystals were then isolated by filtrationand suspended in a 1:1 mixture of acetonitrile-water, filtered again andthen dried under reduced pressure. The dried deferoxamine B free basewas then powdered before being used to prepare the mesylate salt asdescribed in Example 1, last paragraph. The powdered free base was foundto be 97.8% (w/w) pure by HPLC analysis. The mesylate salt obtainedusing this material contained 0.96% impurity (w/w) by HPLC analysis andhad a chloride content of less than 60 ppm.

Example 4

Deferoxamine B mesylate was isolated from crude deferoxamine B accordingto the process of Example 2, except that deferoxamine B hydrochloridewas eluted from the bed of Amberlite® XAD 1180 adsorption resin using15% ethanol-water followed by 25% THF-water. The deferoxamine mesylate(238 g) so obtained was found to have an impurity content of 1.73% (w/w)by HPLC analysis.

Example 5

Crude deferoxamine B hydrochloride was chromatographed according to theprocedure described in Example 1. After the deferoxamine B-containingeluent was concentrated to 90 g L⁻¹ and decolorized as described inExample 1, the decolorized eluent was divided into three equal portionscontaining 40 g of deferoxamine B each. Each portion was diluted with ananti-solvent. One portion was diluted with an equal volume of methanol,another with an equal volume of ethanol and the other with an equalvolume of acetone. The pH of each of the portions was then adjusted tobetween 8.6 and 10.1 with aqueous ammonia causing deferoxamine B freebase to come out of solution. Precipitation was completed by addition oftriple the portion volume of a 1:1 acetonitrile-anti-solvent mixture toeach portion, i.e. either acetonitrile-methanol, acetonitrile-ethanol oracetonitrile-acetone. The suspensions were then cooled to −20 to −5° C.and deferoxamine B free base was isolated from each suspension byfiltration. The crystals were then washed as described in Example 1.Following the procedure for converting the free base to the mesylate inExample 1, deferoxamine B mesylate is obtained in amounts of from 24-28g. starting from 40 g of crude deferoxamine B. The impurity content ofthe so-obtained deferoxamine B mesylate was between 1:1 and 1.5% (w/w).

Example 6

The process of Example 2 was followed, except that the crudedeferoxamine B contained sulfate anion and the Amberlite® XAD 1180 resinwas washed with aqueous ammonium sulfate instead of aqueous ammoniumchloride and then eluted with 10% acetonitrile-water instead of a 1:4methanol-water mixture. The so-obtained deferoxamine B mesylate salt(261 g) had an impurity content of 1.4% (w/w) measured by HPLC.

Example 7

A suspension of deferoxamine B free base was prepared according to theprocedure of Example 2 and cooled as described to 4° C. A one tenthportion of the suspension containing 34.1 g of deferoxamine B free basewas heated to 36° C. and stirred at elevated temperature for 1 hour andthen filtered to recover the free base. The recovered crystals were thendried and converted to the deferoxamine B mesylate as described inExample 2. The product (27 g) had an impurity content of 1.4% (w/w) asdetermined by HPLC analysis.

Example 8

The process of Example 2, paragraphs 1 and 2, was followed, except thata portion of the eluent containing 43.2 g of deferoxamine B was notdecolorized. The free base was twice precipitated from this portion ofthe eluent. The deferoxamine B was first precipitated as described inExample 2, paragraph 2. Then the once-precipitated deferoxamine B basewas suspended in a 1:1 acetonitrile-water mixture at a concentration of40-50 g L⁻¹. One molar hydrochloric acid was then slowly added to thesuspension until the deferoxamine B dissolved, The deferoxamine B freebase was then re-precipitated from the solution by following theprocedure of Example 2, paragraph 2, and was transformed into themesylate salt as described in Example 2, paragraph 3, to yield 27 g ofdeferoxamine B mesylate. Product coloration was indistinguishable fromthat of the product of Example 2. The impurity content as measured byHPLC is 1.4% (w/w).

Example 9

Following the procedure of Example 1, crude deferoxamine B waschromatographed and the eluent containing deferoxamine B (45.3 g) wasdecolorized. The decolorized solution was concentrated to 90 g L⁻¹ andpassed through a bed of Diaion® SP 207 adsorption resin. Proceeding withthe procedure for precipitating the free base and converting it to themesylate salt as described in the second and third paragraphs of Example1, as much as 31.5 g of the mesylate can be obtained and with only 1.46%(w/w) impurity according to HPLC analysis.

Example 10

The procedure of Example 1 was followed except that 200 L ofacetonitrile was substituted for 200 L of ethanol to induceprecipitation of deferoxamine mesylate. Deferoxamine B mesylate wasobtained in 94.7% yield.

Following the procedure of Example 1, but substituting propanol,butanol, amyl alcohol, hexanol and heptanol for ethanol to induceprecipitation of the deferoxamine B mesylate, the mesylate is obtainedin yields ranging from 83.1 to 89.1%.

Substituting ethyl formate, ethyl acetate, butyl acetate, hexane and THFfor ethanol in the procedure of Example 1, deferoxamine B mesylate isobtained in yields ranging from 88.1 to 93.1%.

In each of these variations on the Example 1 procedure, impurity waspresent in less than 2.5% (w/w) as determined by HPLC.

Example 11

Deferoxamine mesylate prepared according to Example 1 was recrystallizedfrom methanol and mixtures of methanol and other solvents in yieldsranging from 81.3 to 93.7%. Recrystallization was performed bydissolving deferoxamine B mesylate in 8.5 volumes of methanol at 35° C.and then adding one of the following solvents at room temperature:methanol, ethanol, propanol, butanol, ethyl formate, ethyl acetate,butyl acetate, hexane, toluene, THF or acetonitrile. The recrystallizeddeferoxamine B mesylate typical had 1.2-1.6% (w/w) impurity, lower thanthe 1.84% (w/w) impurity level of the material before recrystallization.

Having described the invention with reference to its preferredembodiments and having further illustrated the invention with specificexamples, those of ordinary skill in the art may, upon reading thisdisclosure, apprehend modifications that could be made which do notdepart from the spirit and scope of the invention. It should thereforebe understood that the description and examples presented above are forillustrative purposes only and should not be read as limiting the scopeof the invention as set forth in the claims, which follow.

1. A process for isolating deferoxamine B mesylate from a sourcematerial containing deferoxamine B produced by a microbiological processcomprising the steps of: a) adsorbing deferoxamine B onto an adsorptionresin by contacting the source material with the adsorption resin, b)obtaining an eluent containing deferoxamine B by eluting the adsorptionresin having deferoxamine B adsorbed thereon with a mixture of water anda water-soluble organic elution solvent, c) adjusting the pH of theeluent to between about 8.6 and 10.5 with a basic ion exchange resin oran alkaline solution, or both, d) crystallizing deferoxamine B free basefrom the pH-adjusted eluent by addition of a deferoxamine B free baseanti-solvent, optionally after partial concentration of the eluent to adeferoxamine B concentration of not more than 150 g L⁻¹, e) suspendingthe deferoxamine B free base in a mixed solvent comprising adeferoxamine B mesylate anti-solvent and a deferoxamine B mesylatesolvent selected from the group consisting of water and methanol, f)dissolving the deferoxamine B free base in the mixed solvent bycontacting the suspension with methanesulfonic acid, and g)precipitating deferoxamine B mesylate.
 2. The process of claim 1 furthercomprising the preliminary step of passing the source material through apre-column bed of adsorption resin.
 3. The process of claim 1 furthercomprising the preliminary step of adding an inorganic salt to thesource material.
 4. The process of claim 3 wherein the inorganic salt isammonium chloride or ammonium sulfate.
 5. The process of claim 1 whereinthe adsorption resin is eluted with a mixture of water and awater-soluble organic solvent selected from the group consisting ofmethanol, ethanol, acetonitrile and tetrahydrofuran.
 6. The process ofclaim 5 wherein the water-soluble organic solvent is acetonitrile. 7.The process of claim 1 further comprising the intermediate step ofdecolorizing the eluent before adjusting the pH of the eluent.
 8. Theprocess of claim 1 further comprising adjusting the deferoxamine Bconcentration of the eluent before adjusting the pH of the eluent. 9.The process of claim 8 wherein the deferoxamine B concentration in theeluent is adjusted to from about 50 g L⁻¹ to about 150 g L⁻¹ byevaporation.
 10. The process of claim 9 wherein the deferoxamine Bconcentration is adjusted to from about 80 g L⁻¹ to about 100 g L⁻¹. 11.The process of claim 10 wherein the deferoxamine B concentration isadjusted to about 90 g L⁻¹.
 12. The process of claim 8 furthercomprising adding acetonitrile to the eluent afterconcentration-adjustment and before pH-adjustment.
 13. The process ofclaim 1 wherein the pH of the eluent is adjusted to between about 9.4and about
 10. 14. The process of claim 1 where the pH of the eluent isadjusted by addition of an alkaline solution.
 15. The process of claim14 wherein the alkaline solution is a solution of NaOH, KOH, ammonia oran amine.
 16. The process of claim 15 wherein the alkaline solution isaqueous ammonia.
 17. The process of claim 1 wherein the pH of the eluentis adjusted using a basic ion exchange resin.
 18. The process of claim13 wherein the pH of the eluent is adjusted to between about 9.4 andabout 10 by adding a basic ion exchange resin to the eluent until a pHbetween about 8.0 and about 9.3 is reached, separating the basic ionexchange resin and adding aqueous ammonia until a pH of between about9.4 and 10 is reached.
 19. The process of claim 1 wherein the mixedsolvent in which the deferoxamine B free base is suspended compriseswater and a deferoxamine B mesylate anti-solvent selected from the groupconsisting of C₁-C₇ aliphatic alcohols, acetone, methyl formate, methylacetate, ethyl acetate, hexane, toluene, tetrahydrofuran andacetonitrile.
 20. The process of claim 19 wherein the deferoxamine Bmesylate anti-solvent is selected from the group consisting of ethanol,acetone and acetonitrile.
 21. The process of claim 1 wherein the mixedsolvent in which the deferoxamine B free base is suspended comprisesmethanol and a deferoxamine B mesylate anti-solvent selected from thegroup consisting of C₁-C₇ aliphatic alcohols, acetone, methyl formate,methyl acetate, ethyl acetate, hexane, toluene, tetrahydrofuran andacetonitrile.
 22. The process of claim 21 wherein the deferoxamine Bmesylate anti-solvent is selected from the group consisting of ethanol,acetone and acetonitrile.
 23. The process of claim 1 whereinprecipitation of deferoxamine B mesylate is accelerated by cooling. 24.The process of claim 1 wherein precipitation of deferoxamine B mesylateis accelerated by adding a deferoxamine B mesylate anti-solvent selectedfrom the group consisting of C₁-C₇ aliphatic alcohols, acetone, methylformate, methyl acetate, ethyl acetate, hexane, toluene, tetrahydrofuranand acetonitrile.
 25. The process of claim 1 wherein the precipitateddeferoxamine B mesylate has a chloride ion content of about 90 ppm orless.
 26. The process of claim 1 wherein the precipitated deferoxamine Bmesylate contains less than about 2.5 mole % other polyhydroxamatesbased upon the moles of deferoxamine B.